JPH04311399A - Analytic element - Google Patents

Abstract

PURPOSE:To provide an analytic element reduced in undesirable influences due to endogenous creatine in a test solution and excellent in reproducibility and accuracy in measureing an objective test substance. CONSTITUTION:An analytic element for analysis of creatine and/or creatinine in a biological liquid sample, having a reagent layer on a substrate and a developing layer thereon, and containing creatininase in the reagent layer and creatinase in both the reagent layer and the developing layer.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an analytical element for measuring creatine and/or creatinine.

0002

BACKGROUND OF THE INVENTION Analysis of intermediates and end products of protein metabolism is important in clinical chemistry, particularly in the diagnosis of renal function. These metabolic products include creatine and creatinine. Enzymatic analysis of creatinine in the diagnosis of kidney function uses the following reaction pathway that is specific to creatinine and creatine.

0003

[Chemical formula 1]

It has been developed using [0004]. Note that the first reaction is catalyzed by creatinine amide hydrolase (creatininase), and the second reaction is catalyzed by creatine amidinohydrolase (creatinase). However, when measuring creatinine, if the test solution contains creatine, N-ethylglycine, etc. that are catalyzed by creatinase, accurate measurement cannot be performed using an integrated analytical element.

In order to solve this problem, for example, the enzyme creatininase 200-1200I. U. /m2, enzyme creatinase 35000-65000I. U. /m2
, and the enzyme sarcosine oxidase 2000-350
0I. U. /m2 and an absorbent carrier material containing hydrogen peroxide and a leuco dye capable of producing a detectable dye in the presence of a peroxidizing substance, wherein the creatinase is substantially inert to the leuco dye. An analytical element for creatinine or creatine has been proposed in which the creatininase is present in a rate-limiting amount and all three enzymes are present at a pH of about 6.5 to 6.9 (Unexamined Japanese Patent Publication No. Publication No. 63-305254). A specific example is one in which two reagent layers are provided, with the reagent layer closer to the porous developing layer containing creatinase and the reagent layer closer to the support containing creatininase or other enzymes.

However, in this proposal, since there is only one layer containing creatinase, the main reaction and the reaction for eliminating the influence of endogenous creatine must be performed with the same reagent, and the analytical element There is little freedom in design, and furthermore, it is difficult to adjust the reaction balance between the main reaction and endogenous creatine removal, and problems remain in terms of reproducibility and accuracy.

[0007]Also, according to the layer structure described in JP-A No. 64-98952, the influence of the coexisting substance (creatine) can be reduced, but when measuring a sample containing a high concentration of creatine, There are problems in that it is not sufficient, has low sensitivity, and has poor simultaneous reproducibility.

[0008]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide an analytical element in which the adverse effects of endogenous creatine in a test solution are reduced and the measurement of the original test substance is excellent in reproducibility and accuracy. According to the research of the present inventor, it was found that sensitivity increases when creatininase is contained in the reagent layer, creatinase is contained in the development layer, and creatinase is also contained in the reagent layer containing creatininase. .

Furthermore, when sarcosine oxidase is contained in the developing layer, the coexisting substance (creatine) is rapidly decomposed, so it is preferable to remove the influence of the coexisting substance. It was also found that the sensitivity increases when In other words, with such a configuration, endogenous coexisting substances can be reacted and guided to a dye before the creatininase reaction starts. After the coexisting substances have been sufficiently removed and led to the dye, the first reaction, creatininase reaction, is carried out.
By measuring at multiple points (two or more points), it was possible to extremely reduce errors caused by endogenous coexisting substances. However, when creatinase is contained only in the development layer, creatine derived from creatinine by the action of creatininase reacts once with creatinase in the development layer, becomes sarcosine, and then reacts with sarcosine oxidase in the reagent layer. It turns out. For this reason, the reaction took place in the reagent layer - development layer - reagent layer, and did not proceed smoothly. However, by including creatinase in the reagent layer, the reaction in which creatinine is led to the dye becomes smoother. This resulted in increased sensitivity and improved reproducibility.

[0010] The present invention has been achieved based on such knowledge, and the object of the present invention is to detect creatine and/or This is achieved by an analytical element for analyzing creatinine, characterized in that creatininase is contained in a reagent layer, and creatinase is contained in a reagent layer and a developing layer.

[0011] Also, an analytical element for analyzing creatine and/or creatinine in a biological fluid sample, which has a reagent layer on a support and a spreading layer above the reagent layer, wherein creatininase is contained in the reagent layer. This is achieved by an analytical element characterized in that creatinase is contained in a reagent layer and a developing layer, and further, sarcosine oxidase is contained in the developing layer.

[0012] Also, an analytical element for analyzing creatine and/or creatinine in a biological fluid sample, which has a reagent layer on a support and a spreading layer above the reagent layer, wherein creatininase is contained in the reagent layer. This is achieved by an analytical element characterized in that creatinase and sarcosine oxidase are contained in the reagent layer and the developing layer. still,
In the analytical element of the present invention, the content of creatinase is higher in the developing layer than in the reagent layer, especially when the ratio of the creatinase content in the reagent layer to the creatinase content in the developing layer is 1:10 to 100. The ratio of the content of sarcosine oxidase to the content of creatinase is preferably 1:5 to 30.

[0013] Also, the content of creatinase is 25,000
~200000I. U. /m2, and the content of creatininase is preferably 1/2000 to 1/15 of the content of creatinase. A case where the present invention is applied to an analytical element for measuring creatine and/or creatinine will be explained as follows.

Analysis of creatine and/or creatinine is carried out by the following series of reactions (1) to (4).

[0015]

[Case 2]

These reactions are catalyzed by creatininase, creatinase, sarcosine oxidase and peroxidizing substances, respectively. Creatinase and creatinase are commercially available from a number of sources. Several types of each enzyme isolated from various microbial sources are known in the literature. Creatininase with a pH optimum of 6.5 at 37°C and creatinase with a pH optimum of 7.7 at 37°C, both obtained from strains of Flavobacterium, are preferred.

Sarcosine oxidase is also obtained from a number of sources. For example, Japanese Patent Application Laid-Open No. 55-34001,
JP-A-56-92790, JP-A-61-1621
It is described in Publication No. 74. Peroxidizing substances include peroxidases. Peroxidases useful in the present invention are of plant or microbial origin.

The dye precursor used in the present invention can be selected from a group of compounds (one or a combination of two or more compounds) that can be oxidized and detected in the presence of hydrogen peroxide and a peroxidizing substance. For example, 4-aminoantipyrine and its derivatives are described in JP-A-59-46854, leuco dyes are described in JP-A-62-257400, and leuco dyes are described in JP-A-63-127158. Examples include combinations of compounds that

The analytical element of the present invention may contain one or more other additives used in general analytical elements for manufacturing or operational advantages. Such additives include surfactants, ion chelators, buffers, solvents, hardeners, antioxidants, couplers, and the like. Surfactants can be used regardless of whether they are nonionic or ionic. Nonionic surfactants are useful coating aids because they dissolve well in both organic solvents and water, and they also have biological properties. This is particularly preferred since it is effective in improving the development speed when a fluid sample is dropped onto the porous development layer. Specific examples of useful surfactants include Triton sodium triisopropylnaphthalene sulfonate, fluorotenside FT-248 (perfluorooctyl sulfonic acid tetraethylammonium salt, manufactured by Bayer), Florado FC-170C (perfluoroalkyl polyoxyethylene ethanol, 3M Examples include non-ionic and ionic surfactants such as those manufactured by A. In particular, the surfactant contained in the porous spreading layer is preferably a nonionic surfactant, and the following R-O-(CH2-CH2-
O-)n H (wherein R represents a phenyl group substituted with an alkyl group having 1 to 9 carbon atoms or an alkyl group having 1 to 20 carbon atoms, and n represents an integer of 1 to 70) Preferably, it contains 5 to 20 g/m2 of a nonionic surfactant.

[0020] As a buffering agent, please refer to "Chemical Handbook Basic Edition" edited by the Chemical Society of Japan (Tokyo, Maruzen Co., Ltd., published in 1966).
pp. 1312-1320, R. M. C. “Data for Biochemical” edited by Dawson et al.
Research” 2nd edition (Oxford at the Clendon Press, 1969) pp. 476-508, “Biochemistry”
Volume 5, page 467 (1966), “Analyti
104, pages 300-310 (1980).

Specific examples of pH buffers in the range of pH 5.5 to 9.0 include buffers containing tris(hydroxymethyl)aminomethane (Tris), buffers containing phosphate, and borate. Buffers containing citric acid or citrate, buffers containing glycine, N,N-bis(2-hydroxyethyl)glycine [Bicine (Bic
ine)], N-2-hydroxyethylpiperazine-N
'-2-Hydroxypropane-3-sulfonic acid (HEP
PS) Na salt or K salt, etc., N-2-hydroxyethylpiperazine-N'-3-sulfonic acid (EPPS) Na salt or K salt, etc., N-[tris(hydroxymethyl)methyl]
-3-aminopropanesulfonic acid (TAPS) Na salt or K salt, etc., N-2-hydroxyethylpiperazine-N'
-2-ethanesulfonic acid (HEPES) Na salt or K salt, etc., N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) Na salt or K salt, etc., and can be combined with any of these if necessary There are acids, alkalis or salts. Specific examples of preferred buffers include:
Potassium dihydrogen phosphate-disodium hydrogen phosphate, tris-sodium borate, triscitric acid, citric acid-sodium dihydrogen phosphate, bicine, HEPPS, HEPP
S sodium salt, HEPES potassium salt, EPPS, E
Examples include PPS sodium salt, TAPS, TAPS sodium salt, TES, TES sodium salt, TES potassium salt, and the like.

[0022] Since the pH buffer is preferably present in the enzyme-containing layer, it is preferable that the pH buffer is contained in the enzyme-containing porous development layer or reagent layer. Since low molecular weight pH buffers can migrate between layers as water permeates, which is the medium of the applied aqueous liquid sample, pH buffers need to be included in all layers containing the enzyme and coloring reagent compositions. For example, p is only present in the hydrophilic or water absorbing layer.
H buffering agent may be included.

[0023] In the practice of the present invention, incorporating creatinase or sarcosine oxidase into the porous spreading layer can be achieved by many methods. For example, when coating is used as a method for forming a porous development layer, it is common to dissolve it in a solvent of a coating liquid (for example, when the solvent is water). If the solubility of the reagent in the solvent is not appropriate, it may be dispersed in the solvent without being dissolved. Regarding various methods for dispersion, use the methods described in New Experimental Chemistry Course, Volume 18, "Interfaces and Colloids" (Maruzen Co., Ltd., edited by the Chemical Society of Japan, published in 1978), pages 340 to 355. be able to. Moreover, regardless of whether a solvent is used or not,
Creatinase or sarcosine oxidase may be physically or chemically adsorbed to the above-mentioned material constituting the porous developing layer, or may simply be mixed as a part of the material constituting the porous developing layer. Preferably, a nonionic surfactant is added to an organic solvent that is liquid at room temperature, such as benzene, toluene, xylene, ether, hexane, and its derivatives.
A good method is to add the material constituting the porous spreading layer and creatinase or sarcosine oxidase, disperse it using a sand stirrer, homogenizer, ultrasonic homogenizer, etc., and then apply and dry the dispersion to form a porous spreading layer. This is surprising given the fact that soaking enzymes in organic solvents generally reduces enzyme activity. However, from a practical point of view, the time from when the enzyme comes into contact with the organic solvent to when it is applied and dried is preferably within 10 hours, although it varies depending on the type of organic solvent.

Although the porous spreading layer of the dry analytical element of the present invention is self-supporting (ie, composed of a material sufficiently rigid to maintain its original shape), it is preferably formed from another non-porous material. placed on a sexual support. Such a support is of suitable dimension stability, preferably non-porous,
It is a transparent (ie, radiation-transparent) material that is capable of transmitting electromagnetic radiation of wavelengths between 200 and 900 nm. The choice of support for a particular analytical element should be adapted to the mode of detection (transmission or reflection spectrophotometry) to be detected. Useful supports include paper, metal foil, polystyrene, polyester (eg polyethylene terephthalate), polycarbonate or cellulose ester (eg cellulose acetate), and the like.

The porous spreading layer in the present invention is described in US Pat. No. 4,292,272 and US Pat. No. 3,992,158.
specification, U.S. Patent No. 4,258,001, U.S. Patent No. 4,430,436, and JP-A-57-1017
Fibers or non-fibrous substances as described in Japanese Patent Publication No. 61-61347, or a mixture of both.
It is prepared from a fabric such as that described in the publication.

The analytical element of the present invention can have various functional layers and layer configurations as desired. For example, reagent layers, reflective layers, subbing layers as described in US Pat. No. 3,992,158, radiation blocking layers as described in US Pat. No. 4,042,335, barrier layers as described in US Pat. No. 4,066,403, US Pat. No. 4,144,306. Registration layer as described in US Pat. No. 4,166
The migration prevention layer described in No. 093, the scintillation layer described in U.S. Pat. No. 4,127,499, the scavenger layer described in JP-A-55-90859, the breakable pod-shaped member described in U.S. Pat. No. 4,110,079, etc. It is possible to construct a multilayer analytical element that meets the purpose of the present invention by combining them arbitrarily.

The various layers described above can be formed into layers of arbitrary thickness by using slide hopper coating methods, extrusion coating methods, dip coating methods, etc., which are known in the photographic industry. In addition, as for the layer structure, for example, JP-A-51-4
0191 (Japanese Patent Publication No. 58-18628), U.S. Patent No. 4110079, JP-A-58-13156
It is possible to select the layer structure described in Publication No. 5 and the like.

The analytical element manufactured in this way can be made into various shapes depending on the analytical method. For example, it can be of various shapes including tape, sheet or slides mounted on plastic mounts of the desired width. The present invention will be specifically explained below, but the present invention is not limited to this example.

[0029]

[Example] A reagent layer shown in Table 1 below was provided on a transparent undercoated polyethylene terephthalate support having a thickness of about 180 μm, and a vinyl pyrrolidone-vinyl acetate copolymer (polymerization ratio 2:8) was placed on the reagent layer. An adhesive layer (a1) was provided so that the weight was 1.8 g/m2, and a developing layer shown in Table 2 was further provided on this adhesive layer, and analytical elements-1 to -4 of the present invention and comparison shown in Table 3 were prepared. Example analysis elements-1 and -2 were created. Table 1 (Reagent layer) R-1 R-2
R-3 R-4 R-
5 R-6A 20
20 20
20 20
20 B 0.9 0.9
0.9 0.9
0.9 0.9C
1.8 1.8
1.8 1.8
1.8 1.8D 3.
0 3.0 3.0
3.0 3.0
3.0E 0.09
0.09 0.09
0.09 0.09
0.09 F 30000 3000
0 30000 30000
30000 30000G
14000 14000
10000 7000 1
4000-H 6500
6500 6500
6500 6500
6500I 1500 9
000 2000 20
00--J
6.2 6.2
6.2 6.2
6.2 6.2K 1
．． 5 1.5 1.
5 1.5 1.5
1.5L 6.2
6.2 6.2
6.2 6.2
6.2 In Table 1, A is gelatin (g/m2)
, B is sodium triisopropylnaphthalene sulfonate (g/m2), C is dibutyl phthalate (g/m2)
), D is 7-chloro-3-[2-(2-hexyldecylsulfonyl)ethyl]-6-methyl-pyrazolo-[3
,2-c]-s-triazole (g/m2), E is 1,2-bis(vinylsulfonyl)ethane (g/m2)
), F is peroxidase (I.U./m2), G is sarcosine oxidase (I.U./m2), H is creatininase (I.U./m2), and I is creatinase (I.U./m2). /m2), J is N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (
g/m2), K is potassium hydroxide (g/m2), L
is bovine serum albumin (g/m2). Table 2 (Development layer) S-1 S-2
S-3 S-4 S-
5 S-6M 106
106 106
106 106
106 N 11.0 11.
0 11.0 11.0
11.0 11.0
O 12000 12000
12000 12000
12000 12000 P 1125
00 105000 11200
0 112000 114000
114000 Q 11.8
11.8 11.8
11.8 11.8
11.8 R 0.2
0.2 0.2
0.2 0.2
0.2 S 1.7 1
．． 7 1.7 1.
7 1.7 1.7
T--
4000 7000
- 14000 U 2
．． 0 2.0 2.
0 2.0 2.0
2.0 In Table 2, M is fiber for filter paper raw material (g/m2), N is styrene-glycidyl methacrylate copolymer (polymerization ratio 9:1) (g/m2)
, O is ascorbate oxidase (I.U./m2
), P is creatinase (I.U./m2), Q is Triton X-100 (g/m2), R is hydrochloric acid 4-N,N
-diethylamino-2-(2'-methanesulfonamidoethyl) aniline (g/m2), S is 5,5-dimethyl-1,3-cyclohexanedione (g/m2),
T is sarcosine oxidase (I.U./m2), U
is bovine serum albumin (g/m2).

[0030] Moreover, the porous spreading layer is made of Triton X-100
, after dissolving the styrene-glycidyl methacrylate copolymer in 280 g of xylene, adding and stirring the fiber for filter paper raw material and other reagents other than the enzyme, then adding the enzyme,
The resulting dispersion was dispersed with a homogenizer and then applied onto the adhesive layer and dried. Table 3
Reagent layer Adhesive layer
Deployment layer Analytical element-1 of the present invention
R-1 a-1 S-1
Analytical element-2 R-2 of the present invention
a-1 S-2 Analytical element-3 of the present invention-3 R-3 a-
1 S-3 Analytical element-4 of the present invention
R-4 a-1
S-4 Comparative Example Analytical Element-1 R-
5 a-1 S-5
Analytical element-2 R-6 of comparative example
a-1 S-6 For the analytical elements of each example above, creatinine 5.0 mg/dl and creatine 0
．． Sample (1) containing 8 mg/dl and creatinine 5.
Samples (2) containing 0 mg/dl and 6.1 mg/dl of creatine were added dropwise and incubated at 37°C. Then, 546n after 210 seconds and 420 seconds after dropping.
The difference in reflection density at m was converted using a formula created in advance for converting the difference in reflection density into creatinine, and each concentration was determined. Further, the difference in reflection density at 540 nm after 210 seconds of dropping was converted to creatine concentration using a conversion formula created in advance. These results are shown in Table 4. Table 4
Creatinine concentration Creatine concentration
(mg/dl)
(mg/dl)
Sample (1) Sample (2) Sample (1) Sample (2) Analytical element-1 of the present invention
5.0 5.2 0.8
6.1 Analytical element of the present invention-2 5.0
5.2 0.8 6
．． 1 Analytical element of the present invention-3 5.0
5.2 0.8 6.1
Analytical element of the present invention-4 5.0 5.
1 0.8 6.1 Analytical element of comparative example-1 5.1 5.7
0.7 5.9 Analytical element-2 of comparative example 5.0 5.4
0.8 6.1 From Table 4, it can be seen that the analytical element of the present invention has little influence of endogenous creatine in the test substance sample in creatinine measurement.

Furthermore, control serum N "Roche" (manufactured by F. Hoffmann-La Roche) and 1.26 mg of creatinine were added to analytical elements-1 to -4 of the present invention and analytical elements-1 and -2 of comparative examples. Drop 10 μl of dl, incubate at 37°C, determine the difference in reflection density between 3 minutes and 30 seconds and 7 minutes, and convert to a concentration value using a conversion formula created in advance using human serum containing various creatinine. did. This was done for each element with N=18, and the simultaneous reproducibility was investigated.
The results are shown in Table 5. Table 5 (simultaneous reproducibility) Analytical element of the present invention-1 2.1% Analytical element of the present invention-2 3.1%
Analytical element of the present invention-3 2.5%
Analytical element-4 of the present invention
2.9% Analytical element-1 of comparative example
6.9% Comparative Example Analytical Element-2 7.5% From Table 5, it can be seen that the simultaneous reproducibility is improved.

[0032]

[Effects] The analytical element of the present invention is less affected by endogenous creatine, has high sensitivity, and has improved simultaneous reproducibility.

Claims (5)

[Claims] Claim 1: An analytical element for analyzing creatine and/or creatinine in a biological fluid sample, comprising a reagent layer on a support and a spreading layer above the reagent layer, wherein creatininase is contained in the reagent layer. An analytical element characterized in that creatinase is contained in a reagent layer and a developing layer. 2. The analytical element according to claim 1, wherein the content of creatinase is higher in the developing layer than in the reagent layer. [Claim 3] The ratio of the creatinase content in the reagent layer to the creatinase content in the development layer is 1.
The analytical element according to claim 1 or claim 2, characterized in that the number of pairs is 10 to 100. 4. An analytical element for analyzing creatine and/or creatinine in a biological fluid sample, comprising a reagent layer on a support and a spreading layer above the reagent layer, wherein creatininase is contained in the reagent layer. An analytical element characterized in that creatinase is contained in a reagent layer and a developing layer, and sarcosine oxidase is further contained in the developing layer. 5. An analytical element for analyzing creatine and/or creatinine in a biological fluid sample, comprising a reagent layer on a support and a spreading layer above the reagent layer, wherein creatininase is contained in the reagent layer. An analytical element characterized in that creatinase and sarcosine oxidase are contained in the reagent layer and the developing layer.